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Spontaneous contractions of myometrium from humans, non-human primate and rodents are sensitive to selective oxytocin receptor antagonism in vitro
British Journal of Obstetrics and Gynaecology September 2001, Vol. 108, pp. 960±966
Spontaneous contractions of myometrium from humans, nonhuman primate and rodents are sensitive to selective oxytocin receptor antagonism in vitro R. J. Wilson a,*, M. J. Allen a, M. Nandi a, H. Giles a, S. Thornton b Objectives To determine whether: 1. oxytocin receptor antagonists in¯uence spontaneous contractions of myometrium from humans, non-human primates and rodents (in vitro), and 2. vasopressin V1a receptor antagonism is important for inhibition of spontaneous contractions in human myometrium. Design In vitro pharmacology of spontaneous contractions of myometrium from humans and animals. Setting The research laboratories of a university department of obstetrics and gynaecology and a pharmaceutical industry research centre. Interventions Samples of human myometrium were obtained at caesarean section. Tissue strips were suspended in organ baths for isometric force recording. Cumulative concentration effect curves to a selective oxytocin receptor antagonist (L-371,257) and a mixed oxytocin/vasopressin V1a receptor antagonist (atosiban) were obtained. The effect of L-371,257 was also determined in myometrium from non-pregnant rats and marmosets. Main outcome measures The inhibition of spontaneous myometrial contractions in vitro. Results L-371,257 and atosiban signi®cantly inhibited spontaneous activity of human myometrium in a concentration-related manner (P , 0.05), although the effect was more pronounced with L-371,257. Spontaneous contractions of myometrium from non-pregnant rats and marmosets were also inhibited by L-371,257 (atosiban was not tested). Conclusions Spontaneous contractions of myometrium from humans, marmosets and rats are, at least in part, dependent on oxytocin receptor activity, in vitro. L-371,257 and atosiban may be inverse agonists. Selective non-peptide oxytocin receptor antagonists may be effective tocolytics.
INTRODUCTION The use of the cyclic nonapeptide oxytocin as a stimulant of uterine contractions in obstetrics is well documented. Uterine smooth muscle responds with powerful contractions to low concentrations of oxytocin. High af®nity oxytocin receptors in human myometrium increase through gestation 1 and oxytocin receptor mRNA may increase during labour 2. It has been proposed that spontaneous uterine activity during late gestation and at term correlates with the number of oxytocin receptors 3. Indeed, the failure of women to respond to intravenous oxytocin augmentation of labour is associated with reduced oxytocin receptor expression 4. Surprisingly, human labour is not associated with an increase in plasma oxytocin concentration although pulsatile increases in plasma concentration have been detected in some 5 but
a
Receptor Pharmacology Unit, GlaxoSmithKline Medicines Research Centre, Stevenage, UK b Department of Biological Sciences, University of Warwick, Coventry, UK * Correspondence: R. J. Wilson, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. q RCOG 2001 British Journal of Obstetrics and Gynaecology PII: S03 06- 5456(01)0022 6-1
not all 6 studies. The absence of an increase in maternal plasma oxytocin could be explained by local uterine production of the peptide coupled with rapid metabolism. Although an increase in local oxytocin peptide at the onset of labour remains to be demonstrated, synthesis has been inferred on the basis of an increase in decidual mRNA 7,8. Furthermore, oxytocin receptor antagonists appear to reduce uterine activity both in vitro 9 and in vivo 10,11 and thus, a role for oxytocin in human labour is implied. Myometrial strips exhibit spontaneous contractions in vitro which may be analogous to the uterine contractions of labour. The underlying mechanism is unknown although spontaneous activity is reduced by mixed oxytocin/vasopressin (V1a) antagonists 9 in a manner similar to that observed when the peptide oxytocin/V1a receptor antagonist atosiban is administered to labouring women 12±14. Human myometrium is sensitive to vasopressin, an effect mediated by the V1a receptor 15. However, the degree to which oxytocin and V1a receptors contribute to spontaneous contractions of human myometrium is not known. We used the selective non-peptide oxytocin receptor antagonist L-371,257 16 and the mixed oxytocin/V1a receptor antagonist atosiban 17 to assess the role of these receptors in spontaneous contractions of term human myometrium in vitro. The af®nities of www.bjog-elsevier.com
RECEPTOR ANTAGONISM AND SPONTANEOUS UTERINE ACTIVITY 961 Table 1. Af®nity (Ki) values for oxytocin, L-371,257 and atosiban determined in competition radioligand binding experiments at recombinant human oxytocin (h oxytocin) and vasopressin (hV1a) receptors 12,15. Tritiated oxytocin or vasopressin was displaced with unlabelled oxytocin, L-371,257 or atosiban. Af®nity (Ki) values were calculated by application of the Cheng-Prussof 32 correction to displacement curve mid-point concentrations (IC50). Data from Ref. 12 is presented in parentheses. Compound
h oxytocin Ki
Oxytocin L-371,257 Atosiban
1nM (6.8nM) 7.9nM 34nM (397nM)
hV1a Ki 50nM (34.9nM) 794nM 4.0nM (5.7nM)
these antagonists at cloned human receptors expressed in CHO cells 15,18 are shown in Table 1. The effect of L371,257 was also determined in non-pregnant rat and marmoset myometrium. The aims of the present study were: 1. to con®rm and extend the previous observation that oxytocin/V1a antagonism may inhibit spontaneous activity in uterine strips 8 with a structurally unrelated class of oxytocin receptor antagonist; 2. to compare the activity of a selective oxytocin receptor antagonist (L-371,257) with a combined oxytocin/V1a receptor antagonist (atosiban); 3. to investigate the tissue-speci®city of any effect seen; and 4. to investigate whether these effects occurred in other species. METHODS Myometrial biopsies were obtained from the lower uterine segment of women undergoing caesarean section at term, following full written consent. Ethical approval was granted by Cambridge Local Research Ethics Committee (LREC; Dec 89/56). Myometrial tissue was obtained from rats and marmosets post-mortem. Marmosets were killed by anaesthetic overdose (pentobarbitone sodium 120mg/kg as Euthanal) and rats by cervical dislocation according to the requirements of the Animals (Scienti®c Procedures) Act, 1986. Diethylstilbestrol (0.25mg/kg, i.p.) was given to rats 24 hours before death. This treatment has previously been shown to stimulate reproducible spontaneous contractions of rat myometrium 19,20. All samples were collected into Krebs solution (95%O2/5% CO2, pH 7.4) at 48C and used within 48 hours. Strips (10 x 2 x 2mm) of myometrium were mounted in 5ml siliconised organ baths (Linton Instrumentation Ltd, Diss, Norfolk, UK) for isometric force recording. Tissues were suspended in Krebs solution (95% O2/5% CO2, 378C). Changes in force were detected using FT03C force displacement transducers (Grass Instrument Co, Michigan, USA) and recorded digitally on a MacLab data acquisition system running Chart v3.4.2 software (sampling frequency 0.66Hz; AD Instruments, Hastings, East Sussex, UK). Krebs solution was of the following composition: NaCl 118mM, NaHCO3 25mM, KCl q RCOG 2001 Br J Obstet Gynaecol 108, pp. 960±966
4.8mM, KH2PO4 1.2mM, MgSO4 1.2mM, glucose 11.1mM, and CaCl2 1.25mM. Strips were immediately placed under 1g tension and allowed to relax. Fifteen minutes later, tissue tension was mechanically increased to 2g. Tension was again adjusted to 2g seven minutes after this. No further adjustment of tension was performed throughout the duration of the experiment. Spontaneous contractions were established during a subsequent 90-minute equilibration period. Drugs were added either as a single concentration or as increasing cumulative concentrations every 30 minutes. Control strips from the same biopsy were exposed to vehicle alone. Responses of human tissue were normalised against a tonic contraction induced by hypotonic shock (replacement of Krebs solution with distilled water) at the end of the experiment 21. To test for selectivity of action the effect of L-371,257 was also tested against phenylephrine (10mM)-induced contraction of rabbit corpus cavernosum (obtained post-mortem). Data analysis Responses were measured as the area under the forcetime curve (activity integral) in units of gram-seconds. The baseline activity integral (measured between contractions at the start of the experiment) was taken to represent zero while the activity integral generated by hypotonic shock was taken to represent a maximal (100%) level of contractile activity. Because early experiments established that human myometrium did not completely relax between contractions (in contrast to some of our previous ®ndings), the activity integral beneath baseline tone was also included in the measurements. The level of spontaneous contractile activity was then normalised by expressing it as a percentage of that obtained by hypotonic shock. Negative values indicate that tension had fallen below the baseline obtained at the start of the experiment. A four-parameter logistic equation was then ®tted to normalised data in order to estimate concentration-effect curve minima, maxima, mid-points (EC50) and slope parameters (nH) using a computerised non-linear curve-®tting routine (Robosage, GlaxoSmithKline, UK.). A single biopsy provided tissue for a single experiment. Statistical analysis was performed using Spearman's rank correlation coef®cient on full concentration-effect curve data, and one-sided Student's t test on normalised activity integral inhibition at 10mM antagonist. Signi®cance was taken as P , 0.05. Reagents and drugs Krebs solution (10x concentrate) and oxytocin acetate were purchased from Sigma (Poole, UK). Oxytocin was dissolved at 10mM in distilled water, stored at 48C and used within seven days. Antagonists were dissolved in distilled water and stored in aliquots at -208C.
962 R. J. WILSON ET AL.
RESULTS Spontaneous phasic contractions were observed in approximately 80% of human, and in all rat and marmoset myometrial strips. There was a marked variation in the frequency and force of spontaneous contractions in human strips, even in adjacent strips from the same biopsy (Figs. 1 and 2). Both atosiban (10nM±10mM) and L-371,257 (10pM± 10mM) signi®cantly inhibited spontaneous activity in human myometrium in a concentration-dependent manner (Spearman's: P , 0.05)(Fig. 3). The potency and the magnitude of responses to atosiban were lower than those of L-371,257 (Table 2). Total suppression of spontaneous activity was achieved in more L-371,257-treated strips than atosiban-treated strips (Table 3). Furthermore, L-371,257 (but not atosiban) produced decreases in contractile activity below baseline (as de®ned in methods; t test: L-371257 compared with vehicle P , 0.05; atosiban compared with vehicle P . 0.05). In strips where nontotal suppression was not achieved, the decrease elicited by L-371,257 was greater than atosiban. In order to determine whether the effect of L-371,257 was speci®c for human myometrium, the effect on spontaneous activity was investigated in myometrium taken from non-pregnant rats (n 6, Fig. 4) and marmosets (n 2, Fig. 5). Quanti®cation of antagonist effects in these tissues was complicated by changes in baseline tension. In rat myometrium, changes in spontaneous activity could be observed at 10nM L-371,257 in tissues from ®ve out of six animals, and in tissues from all animals at
100nM. Total suppression of spontaneous activity occurred at 100nM L-371,257 in tissues from four out of six animals and in all tissues at 1mM. Spontaneous contractions of marmoset myometrium ceased at 10mM antagonist in tissues from both animals tested. L-371,257 (1mM) had no effect on phenylephrine (10mM)-induced contraction of rabbit corpus cavernosum (n 3, Fig. 6) demonstrating that effects on myometrium were unlikely to be due to non-speci®c smooth muscle relaxation. DISCUSSION We have demonstrated that atosiban and the more speci®c oxytocin receptor antagonist, L-371,257 reduce spontaneous myometrial contractions in vitro. Furthermore, comparison of these two molecules suggests that reduction of spontaneous contractility achieved by a selective non-peptide oxytocin receptor antagonist is more effective than that achieved by a peptide oxytocin/vasopressin V1a receptor antagonist. In human myometrium, both oxytocin and V1a receptors are present and functionally coupled to smooth muscle contraction 22. Since mixed selectivity peptide receptor antagonists bind to both receptors in pregnant human myometrium 23 it is possible that combined antagonism is required for effective tocolysis. In our study, the effective concentrations of L-371,257 are consistent with our published data on the af®nity of the drug for the oxytocin receptor (Ki 7.9nM) and are below the concentrations required for V1a receptor binding
Fig. 1. Recordings from human myometrial strips (prepared from a single biopsy) showing the effects of L-371,257 on spontaneous contractions. Strips were mounted in tissue baths and allowed to develop spontaneous contractions for 90 minutes before vehicle (Veh) or L-371,257 (0.1nM ± 10mM) were added (at mark).
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RECEPTOR ANTAGONISM AND SPONTANEOUS UTERINE ACTIVITY 963
Fig. 2. Typical recordings from human myometrial strips (prepared from a single biopsy) showing the effects of atosiban on spontaneous contractions. Strips were mounted in tissue baths and allowed to develop spontaneous contractions for 90 minutes before vehicle (Veh) or atosiban (10nM±3mM) were added (at mark).
(Ki 794nM) 18. The effect of atosiban was consistent with its lower oxytocin receptor (Ki 34nM) and higher V1a receptor (Ki 4.0nM) af®nity. Although atosiban appears to be effective in clinical studies of threatened preterm labour 12±14, clinical data using the selective oxytocin receptor antagonist L-368,899 24 also suggests that V1a receptor antagonism is not required for tocoly-
sis 25. In fact, selective oxytocin receptor antagonism with L-368,899 not only reversed oxytocin-induced increases in uterine contractility in women post-partum but also caused a reduction in uterine activity below baseline activity in a manner similar to our data with L-371,257 in vitro. It is also interesting to note that the selective nonpeptide V1a receptor antagonist, SR49059, did not produce parallel rightward displacement of vasopressin concentration effect curves, as would be expected for a simple competitive antagonist at the V1a receptor 12. This also suggests that the link between the V1a receptor and Table 2. Mean curve parameters describing the inhibition of spontaneous contractions in human myometrium by L-371,257 and atosiban. Values were obtained by ®tting a four-parameter logistic equation to normalised activity integral data using a computerised curve-®tting routine (Robosage). De®nition of terms: activity integral suppression activity integral in absence of antagonist minus activity integral in the presence of a maximally-effective concentration of antagonist; slope (nH) the Hill coef®cient describing the slope of the ®tted curve; IC50 the concentration of antagonist required to elicit a half-maximal suppression of spontaneous activity. Values are mean [SD] except for IC50 and slope for which geometric mean (95% CI) is quoted. L-371,257
Fig. 3. Effect of cumulative concentrations of vehicle (X), L-371,257 (B) or atosiban (O) on spontaneous contractions in human myometrial tissue. Abscissa shows log molar concentration of antagonist. Vehicle indicates the addition of solute minus antagonist. Ordinate describes spontaneous contractile activity expressed as a percentage of the tonic contraction produced by exposing tissues to water (hypotonic shock). Negative values are generated in instances where reductions in baseline tone are observed. Points are mean (sem) (n 3-7).
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Activity integral in absence of antagonist Activity integral in presence of maximally-effective concentration of antagonist Activity integral suppression Slope (nH; 95% CI) IC50 (95% CI)
Atosiban
7.2 [2.1]
7.6 [1.2]
-8.3 [1.5]
-0.3 [4.2]
15.5 units 0.5 (0.2-0.7) 71 (24-207) pM
7.9 units 0.5 (0.0-0.9) 105 (58-1088) nM
964 R. J. WILSON ET AL. Table 3. The numbers of samples in which a reduction or abolition of spontaneous contractions was obtained as determined by activity integral (AI) data at each antagonist concentration. The numbers are expressed as a cumulative fraction. No. of tissues in which activity integral was reduced by antagonist. Concentration 10pM 100pM 1nM 10nM 100nM
L-371,257 2/7 2/7 4/7 5/7 7/7
Atosiban 0/6 2/6 2/6 4/6 6/6
±±±±±±±±±±±±±±±±±±±±±±±±± 100pM 1nM 10nM 100nM 1mM 10mM Not achieved
No. of tissues in which spontaneous contractions were totally suppressed. 1/7 0/6 1/7 0/6 1/7 1/6 3/7 1/6 4/7 1/6 5/7 2/6 2/7 4/6
myometrial contractility is more complex than the straightforward binding of vasopressin to the V1a receptor 3. Taken together these observations provide support for a principal (though not exclusive) role for the oxytocin receptor in the generation of myometrial contractions. The maximum reduction in activity integral achieved by L-371,257 was greater than that achieved by atosiban in our study. Although we have not examined the basis for this difference directly, the difference in maximum
effect generated may re¯ect pharmacological differences between the two compounds. Alternatively, one may speculate that differences in the physicochemical properties of L-371,257 and atosiban may allow these compounds to access different `pools' of receptors. Indeed, agonists of the angiotensin AT1 receptor 26 have been shown to access functional intracellular receptors and functional proteins for other receptor classes have been found on the nuclear envelope 27. The role of intracellular receptors in relation to the oxytocin receptor system should therefore be examined. The effect of L-371,257 was unlikely to be due to nonspeci®c smooth muscle relaxation (e.g. by blockade of Ltype Ca 21 channels) as there was no effect of L-371,257 on phenylephrine-induced contractions of rabbit corpus cavernosum. This supports the hypothesis that L-371,257 inhibits spontaneous uterine contractions by an action principally at the oxytocin receptor. The effect of L371,257 on spontaneous contractions in both rat and marmoset myometrium suggest that oxytocin receptor activation is also important for spontaneous activity in species other than the human. Since L-371,257 selectively inhibits oxytocin receptor activity, the receptor appears to be fundamental to the generation of spontaneous activity in vitro. The question then arises as to the mechanism of receptor activation. It is now generally recognised that G-protein coupled 7transmembrane receptors can sometimes become activated in the absence of an agonist. This phenomenon is referred to as constitutive receptor activity 28 and is especially likely where high receptor concentrations exist. One explanation is that the high levels of oxytocin recep-
Fig. 4. Recording from rat myometrial strips (from a single animal). Strips were mounted in tissue baths and allowed to develop spontaneous contractions for 90 minutes before vehicle (Veh) or L-371,257 (10nM-10mM) were added (at arrows).
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RECEPTOR ANTAGONISM AND SPONTANEOUS UTERINE ACTIVITY 965
Fig. 5. Typical recording from marmoset myometrial strips (from a single animal). Strips were mounted in tissue baths and allowed to develop spontaneous contractions for 90 minutes before vehicle (Veh) or L-371,257 (0.1, 1 and 10mM) were added (at arrows).
tor expression that exist in late gestation result in receptor activation in the absence of oxytocin, as has recently been reported in mutated oxytocin receptors 29. Receptor activation by constitutive mechanisms is extremely dif®cult to distinguish from activation by endogenous agonist. There is evidence for local formation of oxytocin in some 7 but not all 30 studies. However, continuous autocrine oxytocin synthesis would be required to maintain receptor stimulation in vitro since oxytocin would either diffuse from tissue strips during washing or be degraded by proteolytic enzymes. Constitutive activity may be inhibited by some but not all antagonists. Antagonists which reduce constitutive activity are referred to as inverse agonists. Only a proportion of all antagonists will act as inverse agonists 31 although all prevent agonist-receptor binding. Inverse
Fig. 6. Force recording from strips of rabbit corpus cavernosum. Strips were dissected free from the tunica albugenica and suspended in tissue baths. Tension was induced by 10mM phenylephrine before L-371,257 (10mM) was added.
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agonism is one potential explanation for the production of different clinical effects by receptor antagonists of the same class. We propose that, assuming constitutive activity, L-371,257 and atosiban behave as inverse agonists at the oxytocin receptor in myometrial strips in vitro. It is possible to speculate that, if this phenomenon occurs in vivo, it is important for the development of effective tocolytics since effective medicines would need to prevent constitutive activity. Designing and quantifying the properties of inverse agonists presents formidable challenges for drug discovery. Because constitutively active mutant receptors are generated by substituting one or more selected amino acids for residues not found at those locations in wild type receptors, they may exhibit mutant receptor-speci®c pharmacology and so assay systems using native receptors are being explored. However, construction of assay systems that are reproducibly constitutively active to the same extent as target native systems in both health and disease demands highly re®ned control of receptor expression and transduction. Finally, constitutive activity might have important implications for the mechanism of labour since it could allow labour to be initiated by an increase in oxytocin receptor expression 1 without the need for oxytocin synthesis and release. In conclusion, our results support a principal role for oxytocin receptors in mediating spontaneous contractions of myometrium from human, rat and marmoset in vitro. The mechanism of spontaneous contractility in vitro may
966 R. J. WILSON ET AL.
involve endogenous oxytocin release or constitutive receptor activation. The latter would lead to the proposal that atosiban and L-371,257 are inverse agonists. Finally, assuming that spontaneous contractions in vitro are not dissimilar from uterine contractility in labour, our results suggest that selective oxytocin receptor antagonists may be effective tocolytics.
15.
16.
Acknowledgements
17.
The authors would like to thank the staff of the Receptor Chemistry and Protein Chemistry Units, GlaxoSmithKline, Stevenage for the preparation of L-371,257 and atosiban. Dr S. Thornton was a MRC Clinical Scientist.
18.
References 1. Kimura T, Takemura M, Nomura S, et al. Expression of oxytocin receptor in human pregnant myometrium. Endocrinol 1996;137:780±785. 2. Takemura M, Kimura T, Nomura S, et al. Expression and localization of human oxytocin receptor mRNA and its protein in chorion and decidua during parturition. J Clin Invest 1994;93:2319±2323. 3. Maggi M, Del Carlo P, Fantoni G, et al. Human myometrium during pregnancy contains and responds to V1 vasopressin receptors as well as oxytocin receptors. J Clin Endocrinol Metab 1990;70:1142±1154. 4. Rezapour M, Backstrom T, Ulmsten U. Myometrial steroid concentration and oxytocin receptor density in parturient women at term. Steroids 1996;61:339±344. 5. Fuchs AR, Romero R, Keefe D, Parra M, Oyarzun E, Behnke E. Oxytocin secretion and human parturition: pulse frequency and duration increase during spontaneous labour in women. Am J Obstet Gynecol 1991;165:1515±1523. 6. Thornton S, Davison JM, Baylis PH. Plasma oxytocin during the ®rst and second stages of spontaneous human labour. Acta Endocrinol (Copenh) 1992;126:425±429. 7. Chibbar R, Miller FD, Mitchell BF. Synthesis of oxytocin in amnion, chorion, and decidua may in¯uence the timing of human parturition. J Clin Invest 1993;91:185±192. 8. Miller FD, Chibbar R, Mitchell BF. Synthesis of oxytocin in amnion, chorion and decidua: a potential paracrine role for oxytocin in the onset of human myometrium. Regul Peptides 1993;45:247±251. 9. Kinsler VA, Thornton S, Ashford MLJ, Melin P, Smith SK. The effect of oxytocin antagonists F314 and F792 on the in vitro contractility of human myometrium. Br J Obstet Gynaecol 1996;103:373±376. 10. Goodwin TM, Paul R, Silver H, et al. The effect of the oxytocin antagonist atosiban on preterm uterine activity in the human. Am J Obstet Gynecol 1994;170:474±478. 11. Zeeman GG, Khan-Dawood FS, Dawood MY. Oxytocin and its receptor in pregnancy and parturition: current concepts and clinical implications. Obstet Gynecol 1997;89:873±883. 12. Goodwin TM, Valenzuela G, Silver H, Hayashi R, Creasy GW, Lane R. Treatment of preterm labour with the oxytocin antagonist atosiban. Am J Perinatol 1996;13:143±146. 13. Romero R, Sibai BM, Sanchez-Ramos L, et al. An oxytocin receptor antagonist (atosiban) in the treatment of preterm labor: a randomized, double-blind, placebo-controlled trial with tocolytic rescue. Am J Obstet Gynecol 2000;182:1173±1183. 14. Moutquin JM, Sherman D, Cohen H, et al. Double-blind, randomized, controlled trial of atosiban and ritodrine in the treatment of preterm
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Accepted 21 May 2001
q RCOG 2001 Br J Obstet Gynaecol 108, pp. 960±966
Spontaneous contractions of myometrium from humans, nonhuman primate and rodents are sensitive to selective oxytocin receptor antagonism in vitro R. J. Wilson a,*, M. J. Allen a, M. Nandi a, H. Giles a, S. Thornton b Objectives To determine whether: 1. oxytocin receptor antagonists in¯uence spontaneous contractions of myometrium from humans, non-human primates and rodents (in vitro), and 2. vasopressin V1a receptor antagonism is important for inhibition of spontaneous contractions in human myometrium. Design In vitro pharmacology of spontaneous contractions of myometrium from humans and animals. Setting The research laboratories of a university department of obstetrics and gynaecology and a pharmaceutical industry research centre. Interventions Samples of human myometrium were obtained at caesarean section. Tissue strips were suspended in organ baths for isometric force recording. Cumulative concentration effect curves to a selective oxytocin receptor antagonist (L-371,257) and a mixed oxytocin/vasopressin V1a receptor antagonist (atosiban) were obtained. The effect of L-371,257 was also determined in myometrium from non-pregnant rats and marmosets. Main outcome measures The inhibition of spontaneous myometrial contractions in vitro. Results L-371,257 and atosiban signi®cantly inhibited spontaneous activity of human myometrium in a concentration-related manner (P , 0.05), although the effect was more pronounced with L-371,257. Spontaneous contractions of myometrium from non-pregnant rats and marmosets were also inhibited by L-371,257 (atosiban was not tested). Conclusions Spontaneous contractions of myometrium from humans, marmosets and rats are, at least in part, dependent on oxytocin receptor activity, in vitro. L-371,257 and atosiban may be inverse agonists. Selective non-peptide oxytocin receptor antagonists may be effective tocolytics.
INTRODUCTION The use of the cyclic nonapeptide oxytocin as a stimulant of uterine contractions in obstetrics is well documented. Uterine smooth muscle responds with powerful contractions to low concentrations of oxytocin. High af®nity oxytocin receptors in human myometrium increase through gestation 1 and oxytocin receptor mRNA may increase during labour 2. It has been proposed that spontaneous uterine activity during late gestation and at term correlates with the number of oxytocin receptors 3. Indeed, the failure of women to respond to intravenous oxytocin augmentation of labour is associated with reduced oxytocin receptor expression 4. Surprisingly, human labour is not associated with an increase in plasma oxytocin concentration although pulsatile increases in plasma concentration have been detected in some 5 but
a
Receptor Pharmacology Unit, GlaxoSmithKline Medicines Research Centre, Stevenage, UK b Department of Biological Sciences, University of Warwick, Coventry, UK * Correspondence: R. J. Wilson, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. q RCOG 2001 British Journal of Obstetrics and Gynaecology PII: S03 06- 5456(01)0022 6-1
not all 6 studies. The absence of an increase in maternal plasma oxytocin could be explained by local uterine production of the peptide coupled with rapid metabolism. Although an increase in local oxytocin peptide at the onset of labour remains to be demonstrated, synthesis has been inferred on the basis of an increase in decidual mRNA 7,8. Furthermore, oxytocin receptor antagonists appear to reduce uterine activity both in vitro 9 and in vivo 10,11 and thus, a role for oxytocin in human labour is implied. Myometrial strips exhibit spontaneous contractions in vitro which may be analogous to the uterine contractions of labour. The underlying mechanism is unknown although spontaneous activity is reduced by mixed oxytocin/vasopressin (V1a) antagonists 9 in a manner similar to that observed when the peptide oxytocin/V1a receptor antagonist atosiban is administered to labouring women 12±14. Human myometrium is sensitive to vasopressin, an effect mediated by the V1a receptor 15. However, the degree to which oxytocin and V1a receptors contribute to spontaneous contractions of human myometrium is not known. We used the selective non-peptide oxytocin receptor antagonist L-371,257 16 and the mixed oxytocin/V1a receptor antagonist atosiban 17 to assess the role of these receptors in spontaneous contractions of term human myometrium in vitro. The af®nities of www.bjog-elsevier.com
RECEPTOR ANTAGONISM AND SPONTANEOUS UTERINE ACTIVITY 961 Table 1. Af®nity (Ki) values for oxytocin, L-371,257 and atosiban determined in competition radioligand binding experiments at recombinant human oxytocin (h oxytocin) and vasopressin (hV1a) receptors 12,15. Tritiated oxytocin or vasopressin was displaced with unlabelled oxytocin, L-371,257 or atosiban. Af®nity (Ki) values were calculated by application of the Cheng-Prussof 32 correction to displacement curve mid-point concentrations (IC50). Data from Ref. 12 is presented in parentheses. Compound
h oxytocin Ki
Oxytocin L-371,257 Atosiban
1nM (6.8nM) 7.9nM 34nM (397nM)
hV1a Ki 50nM (34.9nM) 794nM 4.0nM (5.7nM)
these antagonists at cloned human receptors expressed in CHO cells 15,18 are shown in Table 1. The effect of L371,257 was also determined in non-pregnant rat and marmoset myometrium. The aims of the present study were: 1. to con®rm and extend the previous observation that oxytocin/V1a antagonism may inhibit spontaneous activity in uterine strips 8 with a structurally unrelated class of oxytocin receptor antagonist; 2. to compare the activity of a selective oxytocin receptor antagonist (L-371,257) with a combined oxytocin/V1a receptor antagonist (atosiban); 3. to investigate the tissue-speci®city of any effect seen; and 4. to investigate whether these effects occurred in other species. METHODS Myometrial biopsies were obtained from the lower uterine segment of women undergoing caesarean section at term, following full written consent. Ethical approval was granted by Cambridge Local Research Ethics Committee (LREC; Dec 89/56). Myometrial tissue was obtained from rats and marmosets post-mortem. Marmosets were killed by anaesthetic overdose (pentobarbitone sodium 120mg/kg as Euthanal) and rats by cervical dislocation according to the requirements of the Animals (Scienti®c Procedures) Act, 1986. Diethylstilbestrol (0.25mg/kg, i.p.) was given to rats 24 hours before death. This treatment has previously been shown to stimulate reproducible spontaneous contractions of rat myometrium 19,20. All samples were collected into Krebs solution (95%O2/5% CO2, pH 7.4) at 48C and used within 48 hours. Strips (10 x 2 x 2mm) of myometrium were mounted in 5ml siliconised organ baths (Linton Instrumentation Ltd, Diss, Norfolk, UK) for isometric force recording. Tissues were suspended in Krebs solution (95% O2/5% CO2, 378C). Changes in force were detected using FT03C force displacement transducers (Grass Instrument Co, Michigan, USA) and recorded digitally on a MacLab data acquisition system running Chart v3.4.2 software (sampling frequency 0.66Hz; AD Instruments, Hastings, East Sussex, UK). Krebs solution was of the following composition: NaCl 118mM, NaHCO3 25mM, KCl q RCOG 2001 Br J Obstet Gynaecol 108, pp. 960±966
4.8mM, KH2PO4 1.2mM, MgSO4 1.2mM, glucose 11.1mM, and CaCl2 1.25mM. Strips were immediately placed under 1g tension and allowed to relax. Fifteen minutes later, tissue tension was mechanically increased to 2g. Tension was again adjusted to 2g seven minutes after this. No further adjustment of tension was performed throughout the duration of the experiment. Spontaneous contractions were established during a subsequent 90-minute equilibration period. Drugs were added either as a single concentration or as increasing cumulative concentrations every 30 minutes. Control strips from the same biopsy were exposed to vehicle alone. Responses of human tissue were normalised against a tonic contraction induced by hypotonic shock (replacement of Krebs solution with distilled water) at the end of the experiment 21. To test for selectivity of action the effect of L-371,257 was also tested against phenylephrine (10mM)-induced contraction of rabbit corpus cavernosum (obtained post-mortem). Data analysis Responses were measured as the area under the forcetime curve (activity integral) in units of gram-seconds. The baseline activity integral (measured between contractions at the start of the experiment) was taken to represent zero while the activity integral generated by hypotonic shock was taken to represent a maximal (100%) level of contractile activity. Because early experiments established that human myometrium did not completely relax between contractions (in contrast to some of our previous ®ndings), the activity integral beneath baseline tone was also included in the measurements. The level of spontaneous contractile activity was then normalised by expressing it as a percentage of that obtained by hypotonic shock. Negative values indicate that tension had fallen below the baseline obtained at the start of the experiment. A four-parameter logistic equation was then ®tted to normalised data in order to estimate concentration-effect curve minima, maxima, mid-points (EC50) and slope parameters (nH) using a computerised non-linear curve-®tting routine (Robosage, GlaxoSmithKline, UK.). A single biopsy provided tissue for a single experiment. Statistical analysis was performed using Spearman's rank correlation coef®cient on full concentration-effect curve data, and one-sided Student's t test on normalised activity integral inhibition at 10mM antagonist. Signi®cance was taken as P , 0.05. Reagents and drugs Krebs solution (10x concentrate) and oxytocin acetate were purchased from Sigma (Poole, UK). Oxytocin was dissolved at 10mM in distilled water, stored at 48C and used within seven days. Antagonists were dissolved in distilled water and stored in aliquots at -208C.
962 R. J. WILSON ET AL.
RESULTS Spontaneous phasic contractions were observed in approximately 80% of human, and in all rat and marmoset myometrial strips. There was a marked variation in the frequency and force of spontaneous contractions in human strips, even in adjacent strips from the same biopsy (Figs. 1 and 2). Both atosiban (10nM±10mM) and L-371,257 (10pM± 10mM) signi®cantly inhibited spontaneous activity in human myometrium in a concentration-dependent manner (Spearman's: P , 0.05)(Fig. 3). The potency and the magnitude of responses to atosiban were lower than those of L-371,257 (Table 2). Total suppression of spontaneous activity was achieved in more L-371,257-treated strips than atosiban-treated strips (Table 3). Furthermore, L-371,257 (but not atosiban) produced decreases in contractile activity below baseline (as de®ned in methods; t test: L-371257 compared with vehicle P , 0.05; atosiban compared with vehicle P . 0.05). In strips where nontotal suppression was not achieved, the decrease elicited by L-371,257 was greater than atosiban. In order to determine whether the effect of L-371,257 was speci®c for human myometrium, the effect on spontaneous activity was investigated in myometrium taken from non-pregnant rats (n 6, Fig. 4) and marmosets (n 2, Fig. 5). Quanti®cation of antagonist effects in these tissues was complicated by changes in baseline tension. In rat myometrium, changes in spontaneous activity could be observed at 10nM L-371,257 in tissues from ®ve out of six animals, and in tissues from all animals at
100nM. Total suppression of spontaneous activity occurred at 100nM L-371,257 in tissues from four out of six animals and in all tissues at 1mM. Spontaneous contractions of marmoset myometrium ceased at 10mM antagonist in tissues from both animals tested. L-371,257 (1mM) had no effect on phenylephrine (10mM)-induced contraction of rabbit corpus cavernosum (n 3, Fig. 6) demonstrating that effects on myometrium were unlikely to be due to non-speci®c smooth muscle relaxation. DISCUSSION We have demonstrated that atosiban and the more speci®c oxytocin receptor antagonist, L-371,257 reduce spontaneous myometrial contractions in vitro. Furthermore, comparison of these two molecules suggests that reduction of spontaneous contractility achieved by a selective non-peptide oxytocin receptor antagonist is more effective than that achieved by a peptide oxytocin/vasopressin V1a receptor antagonist. In human myometrium, both oxytocin and V1a receptors are present and functionally coupled to smooth muscle contraction 22. Since mixed selectivity peptide receptor antagonists bind to both receptors in pregnant human myometrium 23 it is possible that combined antagonism is required for effective tocolysis. In our study, the effective concentrations of L-371,257 are consistent with our published data on the af®nity of the drug for the oxytocin receptor (Ki 7.9nM) and are below the concentrations required for V1a receptor binding
Fig. 1. Recordings from human myometrial strips (prepared from a single biopsy) showing the effects of L-371,257 on spontaneous contractions. Strips were mounted in tissue baths and allowed to develop spontaneous contractions for 90 minutes before vehicle (Veh) or L-371,257 (0.1nM ± 10mM) were added (at mark).
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RECEPTOR ANTAGONISM AND SPONTANEOUS UTERINE ACTIVITY 963
Fig. 2. Typical recordings from human myometrial strips (prepared from a single biopsy) showing the effects of atosiban on spontaneous contractions. Strips were mounted in tissue baths and allowed to develop spontaneous contractions for 90 minutes before vehicle (Veh) or atosiban (10nM±3mM) were added (at mark).
(Ki 794nM) 18. The effect of atosiban was consistent with its lower oxytocin receptor (Ki 34nM) and higher V1a receptor (Ki 4.0nM) af®nity. Although atosiban appears to be effective in clinical studies of threatened preterm labour 12±14, clinical data using the selective oxytocin receptor antagonist L-368,899 24 also suggests that V1a receptor antagonism is not required for tocoly-
sis 25. In fact, selective oxytocin receptor antagonism with L-368,899 not only reversed oxytocin-induced increases in uterine contractility in women post-partum but also caused a reduction in uterine activity below baseline activity in a manner similar to our data with L-371,257 in vitro. It is also interesting to note that the selective nonpeptide V1a receptor antagonist, SR49059, did not produce parallel rightward displacement of vasopressin concentration effect curves, as would be expected for a simple competitive antagonist at the V1a receptor 12. This also suggests that the link between the V1a receptor and Table 2. Mean curve parameters describing the inhibition of spontaneous contractions in human myometrium by L-371,257 and atosiban. Values were obtained by ®tting a four-parameter logistic equation to normalised activity integral data using a computerised curve-®tting routine (Robosage). De®nition of terms: activity integral suppression activity integral in absence of antagonist minus activity integral in the presence of a maximally-effective concentration of antagonist; slope (nH) the Hill coef®cient describing the slope of the ®tted curve; IC50 the concentration of antagonist required to elicit a half-maximal suppression of spontaneous activity. Values are mean [SD] except for IC50 and slope for which geometric mean (95% CI) is quoted. L-371,257
Fig. 3. Effect of cumulative concentrations of vehicle (X), L-371,257 (B) or atosiban (O) on spontaneous contractions in human myometrial tissue. Abscissa shows log molar concentration of antagonist. Vehicle indicates the addition of solute minus antagonist. Ordinate describes spontaneous contractile activity expressed as a percentage of the tonic contraction produced by exposing tissues to water (hypotonic shock). Negative values are generated in instances where reductions in baseline tone are observed. Points are mean (sem) (n 3-7).
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Activity integral in absence of antagonist Activity integral in presence of maximally-effective concentration of antagonist Activity integral suppression Slope (nH; 95% CI) IC50 (95% CI)
Atosiban
7.2 [2.1]
7.6 [1.2]
-8.3 [1.5]
-0.3 [4.2]
15.5 units 0.5 (0.2-0.7) 71 (24-207) pM
7.9 units 0.5 (0.0-0.9) 105 (58-1088) nM
964 R. J. WILSON ET AL. Table 3. The numbers of samples in which a reduction or abolition of spontaneous contractions was obtained as determined by activity integral (AI) data at each antagonist concentration. The numbers are expressed as a cumulative fraction. No. of tissues in which activity integral was reduced by antagonist. Concentration 10pM 100pM 1nM 10nM 100nM
L-371,257 2/7 2/7 4/7 5/7 7/7
Atosiban 0/6 2/6 2/6 4/6 6/6
±±±±±±±±±±±±±±±±±±±±±±±±± 100pM 1nM 10nM 100nM 1mM 10mM Not achieved
No. of tissues in which spontaneous contractions were totally suppressed. 1/7 0/6 1/7 0/6 1/7 1/6 3/7 1/6 4/7 1/6 5/7 2/6 2/7 4/6
myometrial contractility is more complex than the straightforward binding of vasopressin to the V1a receptor 3. Taken together these observations provide support for a principal (though not exclusive) role for the oxytocin receptor in the generation of myometrial contractions. The maximum reduction in activity integral achieved by L-371,257 was greater than that achieved by atosiban in our study. Although we have not examined the basis for this difference directly, the difference in maximum
effect generated may re¯ect pharmacological differences between the two compounds. Alternatively, one may speculate that differences in the physicochemical properties of L-371,257 and atosiban may allow these compounds to access different `pools' of receptors. Indeed, agonists of the angiotensin AT1 receptor 26 have been shown to access functional intracellular receptors and functional proteins for other receptor classes have been found on the nuclear envelope 27. The role of intracellular receptors in relation to the oxytocin receptor system should therefore be examined. The effect of L-371,257 was unlikely to be due to nonspeci®c smooth muscle relaxation (e.g. by blockade of Ltype Ca 21 channels) as there was no effect of L-371,257 on phenylephrine-induced contractions of rabbit corpus cavernosum. This supports the hypothesis that L-371,257 inhibits spontaneous uterine contractions by an action principally at the oxytocin receptor. The effect of L371,257 on spontaneous contractions in both rat and marmoset myometrium suggest that oxytocin receptor activation is also important for spontaneous activity in species other than the human. Since L-371,257 selectively inhibits oxytocin receptor activity, the receptor appears to be fundamental to the generation of spontaneous activity in vitro. The question then arises as to the mechanism of receptor activation. It is now generally recognised that G-protein coupled 7transmembrane receptors can sometimes become activated in the absence of an agonist. This phenomenon is referred to as constitutive receptor activity 28 and is especially likely where high receptor concentrations exist. One explanation is that the high levels of oxytocin recep-
Fig. 4. Recording from rat myometrial strips (from a single animal). Strips were mounted in tissue baths and allowed to develop spontaneous contractions for 90 minutes before vehicle (Veh) or L-371,257 (10nM-10mM) were added (at arrows).
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RECEPTOR ANTAGONISM AND SPONTANEOUS UTERINE ACTIVITY 965
Fig. 5. Typical recording from marmoset myometrial strips (from a single animal). Strips were mounted in tissue baths and allowed to develop spontaneous contractions for 90 minutes before vehicle (Veh) or L-371,257 (0.1, 1 and 10mM) were added (at arrows).
tor expression that exist in late gestation result in receptor activation in the absence of oxytocin, as has recently been reported in mutated oxytocin receptors 29. Receptor activation by constitutive mechanisms is extremely dif®cult to distinguish from activation by endogenous agonist. There is evidence for local formation of oxytocin in some 7 but not all 30 studies. However, continuous autocrine oxytocin synthesis would be required to maintain receptor stimulation in vitro since oxytocin would either diffuse from tissue strips during washing or be degraded by proteolytic enzymes. Constitutive activity may be inhibited by some but not all antagonists. Antagonists which reduce constitutive activity are referred to as inverse agonists. Only a proportion of all antagonists will act as inverse agonists 31 although all prevent agonist-receptor binding. Inverse
Fig. 6. Force recording from strips of rabbit corpus cavernosum. Strips were dissected free from the tunica albugenica and suspended in tissue baths. Tension was induced by 10mM phenylephrine before L-371,257 (10mM) was added.
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agonism is one potential explanation for the production of different clinical effects by receptor antagonists of the same class. We propose that, assuming constitutive activity, L-371,257 and atosiban behave as inverse agonists at the oxytocin receptor in myometrial strips in vitro. It is possible to speculate that, if this phenomenon occurs in vivo, it is important for the development of effective tocolytics since effective medicines would need to prevent constitutive activity. Designing and quantifying the properties of inverse agonists presents formidable challenges for drug discovery. Because constitutively active mutant receptors are generated by substituting one or more selected amino acids for residues not found at those locations in wild type receptors, they may exhibit mutant receptor-speci®c pharmacology and so assay systems using native receptors are being explored. However, construction of assay systems that are reproducibly constitutively active to the same extent as target native systems in both health and disease demands highly re®ned control of receptor expression and transduction. Finally, constitutive activity might have important implications for the mechanism of labour since it could allow labour to be initiated by an increase in oxytocin receptor expression 1 without the need for oxytocin synthesis and release. In conclusion, our results support a principal role for oxytocin receptors in mediating spontaneous contractions of myometrium from human, rat and marmoset in vitro. The mechanism of spontaneous contractility in vitro may
966 R. J. WILSON ET AL.
involve endogenous oxytocin release or constitutive receptor activation. The latter would lead to the proposal that atosiban and L-371,257 are inverse agonists. Finally, assuming that spontaneous contractions in vitro are not dissimilar from uterine contractility in labour, our results suggest that selective oxytocin receptor antagonists may be effective tocolytics.
15.
16.
Acknowledgements
17.
The authors would like to thank the staff of the Receptor Chemistry and Protein Chemistry Units, GlaxoSmithKline, Stevenage for the preparation of L-371,257 and atosiban. Dr S. Thornton was a MRC Clinical Scientist.
18.
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Accepted 21 May 2001
q RCOG 2001 Br J Obstet Gynaecol 108, pp. 960±966